A Sensitive Chemotaxis Assay Using a Novel Microfluidic Device
Figure 3
Cell migration in scratch and Boyden chamber assays. (a) Mitomycin-C (MMC) inhibition of SMC proliferation. SMC treated with or without MMC (40 μg/mL for 30 min at 37°C) were plated in 6-well plates and subsequently harvested. Cell counts were manually performed on a hemocytometer after 1 h, 24 h, and 48 h; cell counts are expressed as the mean ± one standard deviation of triplicates. Trypan blue exclusion showed no differences in cell viability over 48 hr (%). MMC treatment leads to stable numbers of cells collected at 24 h and 48 h with significantly increased proliferation in the non-MMC-treated populations (). (b) Scratch assay; no differences are seen in the extent of migration between cells cultured in no chemokine versus 25–100 ng/mL PDGF; over the 3–6 hours of this assay, no significant difference is seen between control cells and those treated with MMC. (c) Transwell assay; different concentrations of PDGF were added to the lower chamber of transwell devices at time zero; cells on the lower aspect of the transwell insert were enumerated after 6 h, 12 h, or 24 h. Compared with no PDGF in the lower chamber, there were significantly greater cell numbers of migrated cells in 10 ng/mL, 25 ng/mL, and 50 ng/mL PDGF group after 12 h (). After 24 hours, migrated cell numbers in transwells with 5–50 ng/mL PDGF were not significantly different relative to chambers without PDGF. After 24 hours, migrated cell numbers when 100 ng/mL PDGF was present in the lower chamber were significantly reduced relative to the control 0 ng/mL PDGF group. (d) Chemokinesis in transwells; the same 50 ng/mL concentration of PDGF was loaded into bottom chamber, top chamber, or both bottom and top chambers; cell migration was analyzed after 6 h, 12 h, or 24 h. Relative to no PDGF in either chamber, cell migration was significantly greater with 50 ng/mL in the lower chamber at 12 hours (); by 24 hours, there was no significant difference between control chambers and 50 ng/mL PDGF. Notably, adding PDGF to the upper chamber, with or without PDGF in the lower chamber, led to significantly increased migration at 12 hours relative to PDGF in the lower chamber alone ().